Heretofore, biological tissues when frozen developed ice crystals large enough to destroy cellular function and impede viewing by electron microscopy. This could be avoided only where the tissue was either extremely small, or prepared in special cryoprotective media. Various devices developed in an attempt to freeze portions of large tissues without cryoprotective agents included: metal anvils cooled to cryogenic temperatures and brought into contact with tissue surfaces; gaseous sprays; carbon dioxide sprays in pressurized liquid form or in solid form; and sprays of liquified nitrogen, oxygen, or helium. While liquid nitrogen and liquid helium are considered standard in the art for achieving low temperatures conveniently, they do not cool as rapidly as generally expected, since they form an envelope of gaseous insulation immediately surrounding the specimen surface. In like manner, fluid baths simply brought in contact with surfaces can have a barrier layer impeding cooling, as taught by Alger et. al. (U.S. Pat. No. 4,068,495). For very small objects such as individual seeds or blood cells, direct cooling in quenchant liquids held near their melting temperature has been the most successful technique for rapid freezing.
The use of chemical agents including fixatives and chemical media can distort the normal spatial relationships between cellular and tissue components, and can allow the diffusion of substances for a period of many minutes before fixation can occur. Even after conventional means of fixation, small ionic forms can diffuse readily and be lost to analysis.